The goal of this proposal is to genetically identify and biochemically characterize components that mediate intraorganellar protein sorting and translocation to the thylakoid membrane in maize chloroplasts. Two distinct systems that target proteins across the thylakoid membrane have been described. One pathway, the delta pH translocase, is driven solely by a proton motive force and is related to a recently discovered Sec-independent export mechanism in bacteria (the "Tat" translocase). This translocase now appears to be universal in eubacteria; a homologue of one component of this pathway has also been found in plant mitochondria, suggesting that a related mechanism may also exist in all mitochondria. This translocation system is of special interest because of its unusual, and still mysterious, properties: no nucleotide hydrolysis is required and this pathway can accommodate tightly folded proteins while at the same time maintaining the transthylakoid delta pH. However, we still do not know how many proteins comprise this translocase, or the mechanisms by which substrate proteins are recognized and translocated across the membrane. The second system, the cpSec pathway, requires ATP hydrolysis and is homologous to the secretory (Sec) system, but some evidence suggests that chloroplast-specific factors may also be required. My host laboratory has genetically identified new components of both pathways, and my goal is to clone these genes and study the interactions of their gene products with previously identified proteins. This unique collection of targeting mutants will also allow me to study another question that has yet to be adequately addressed: what are the mechanisms by which chloroplast-encoded proteins are targeted to the thylakoid membrane.